PROJECTSUMMARY?PROJECT3 Theextraordinaryanddurablepatientresponseobservedinrecentimmunotherapytrialshavecatapultedtherole of immunosuppressive checkpoints to the forefront of mechanisms of disease maintenance and treatment response. On the wake of this progress, there have been renewed efforts to map the cellular and signaling requirements of local immunosuppression with the goal to expand immunotherapy options to common malignancies,includingprostatecancer.Inthiscontext,anabnormaldifferentiationofmyeloidcellsisoneofthe major immunological hallmarks of cancer. This reflects the expansion of pathologically activated immature myeloid cells, Myeloid-Derived Suppressor Cells (MDSC) with the ability to suppress a variety of immune functionsintumors,andlimittheefficacyofimmunotherapy.Despitetheirimportanceintumormaintenance,and aspotentialdriversofmetastaticcompetency,therearestilllargegapsinourunderstandingofMDSCfunctions. Howthesecellsaccumulateinprimaryaswellasmetastaticsites,potentiallycontributingtoametastaticniche,is poorly understood, and their metabolic requirements have not been explored. In prostate cancer, the role of MDSC is unknown. The present application is designed to fill this knowledge gap and explore an innovative hypothesis for MDSC function. We propose that mitochondrial metabolic reprogramming critically regulates the immunosuppressive checkpoints maintained by MDSC in the prostate cancer microenvironment, ultimately contributingtodiseaseprogressionandincreasedmetastaticcompetency.Consistentwiththismodel,ourrecent results have shown that MDSC have more mitochondria than mature neutrophils or monocytes, and thus produce more ATP, which in turn supports increased cell motility and homing to primary or metastatic tumor sites. Biochemically, we have shown that this pathway involves deregulated production of reactive oxygen species (ROS) via upregulation of cytoplasmic NADPH oxidase, increased oxidation of proteins and phospholipids, and ultimately damage of the mitochondrial outer membrane resulting in organelle dysfunction andcelldeath.WealsosuggestthatmitochondrialdamageprogressivelyaccumulatesduringMDSCmigration fromthebonemarrow(whereitisminimal)totissues(whereitbecomesextensive).Inthefirstspecificaim,we will characterize the mechanism of increased MDSC migration in prostate cancer and test the role of mitochondrialmetabolicreprogrammingandcellularrespirationinthisresponse.Thesecondspecificaimwewill testtheconceptthatmitochondrialdamageinMDSCisaccumulatedwithaprecise,developmentallyregulated timing during migration of these cells from the bone marrow to peripheral tissues. The third specific aim will dissect the hypothesis that damaged mitochondria contribute to MDSC-mediated immunosuppression, and evaluate novel therapeutic strategy targeting mitochondria in MDSC. Synergistically with the other Projects on this P01 application, Project 3 is designed to elucidate novel mechanistic requirements of local immunosuppressionthatmaypromoteprostatecancerprogressiontoanincurable,metastaticstage.Theresults willuncovernovelvulnerabilitiesofMDSCsuitablefortherapeuticinterventioninpatientswithadvancedprostate cancer.